Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

In Vivo Observations of Myosin II Dynamics Support a Role in Rear RetractionV⃞

To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cells also exhibited mechanical defects in rotation, because they still moved aberrantly when seeded into wild-type mounds with proper rotational guidance cues. The mechanical defects in rotation may be mediated by the C-to-spot, because RLC− cells exhibited a defective C-to-spot, including a slower C-to-spot transition, consistent with this mutant’s slower rotational velocity.

Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.